Structure and function of Semaphorin-5A glycosaminoglycan interactions

Integration of extracellular signals by neurons is pivotal for brain development, plasticity, and repair. Axon guidance relies on receptor-ligand interactions crosstalking with extracellular matrix components. Semaphorin-5A (Sema5A) is a bifunctional guidance cue exerting attractive and inhibitory effects on neuronal growth through the interaction with heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycans (GAGs), respectively. Sema5A harbors seven thrombospondin type-1 repeats (TSR1-7) important for GAG binding, however the underlying molecular basis and functions in vivo remain enigmatic. Here we dissect the structural basis for Sema5A:GAG specificity and demonstrate the functional significance of this interaction in vivo. Using x-ray crystallography, we reveal a dimeric fold variation for TSR4 that accommodates GAG interactions. TSR4 co-crystal structures identify binding residues validated by site-directed mutagenesis. In vitro and cell-based assays uncover specific GAG epitopes necessary for TSR association. We demonstrate that HS-GAG binding is preferred over CS-GAG and mediates Sema5A oligomerization. In vivo, Sema5A:GAG interactions are necessary for Sema5A function and regulate Plexin-A2 dependent dentate progenitor cell migration. Our study rationalizes Sema5A associated developmental and neurological disorders and provides mechanistic insights into how multifaceted guidance functions of a single transmembrane cue are regulated by proteoglycans.

a-e Heparin affinity chromatography of Sema5A constructs.Purified proteins of a, Sema5Asema-TSR1-7, b, Sema5Asema-TSR2, c, Sema5ATSR3-4 R747E/R749E, d, Sema5ATSR3-4 K734E/R747E/R749E and e, Sema5Asema-TSR1-7 R747E/R749E were subjected to a heparin affinity chromatography assay.Protein elution was followed by UV absorption at 280 nm (blue line) and the elution gradient is represented by the conductivity trace (orange line).Sema5Asema-TSR1-7 was eluted with ~ 490 mM NaCl, 10 mM HEPES pH 7.5 (conductivity 45 mS/cm at the peak), whereas the other constructs did not bind to the column and was eluted with the loading buffer (∼17 mS/cm).f-h Biolayer interferometry titration sensorgrams for characterization of Sema5A sema-TSR1-7 interaction with glycosaminoglycans; f, Sema5A WT with heparin, g, Sema5A WT with CS-E, h, Sema5A R747E,R749E with CS-E and i, Sema5A WT with CS-A.j, Western blot confirming biotinylation of Sema5A TSR3-4 and its mutants used for the GAGOme assay.Avi-tag biotinylated samples of 1: Sema5A TSR3-4-3C-Avi-His6 wild type and its mutants 2: R747E/R749E and 3: K734E/R747E/R749E were loaded on SDS-PAGE under non-reducing and reducing conditions.Note the decrease in the apparent mass of sample bands under reducing conditions, consistent with the disulfide-linked dimer architecture of the construct.The blot was incubated with Streptactin HRP (BioRad) antibody at 1:25,000 dilution.k, A subset of the genes controlling HS and CS chain elongation and modifications that were engineered with KO (red star) or KI (blue star) in CHO cells and used in this study.l, Representative example (CHO WT cells) of the flow cytometry gating strategy used for dissection of Sema5ATSR3-4 binding to GAGOme cell lines.Intact cells were gated as shown by plotting forward scatter area versus side scatter area.From the intact cell population, single cells were gated as shown by plotting forward scatter width versus forward scatter height.Mean fluorescence intensity (MFI) values for Alexa Fluor 488 was then determined for the intact singlet cells.All flow cytometry experiments were performed using the gating strategy shown here.m, Sema5ATSR3-4 binding to genetically engineered CHO cell lines reveals GAG specificity of Sema5A.Source data are provided as a Source Data file.a, Anomalous difference Fourier map (magenta mesh) calculated for the Sema5ATSR3-4-SO4 dimer in the crystallographic asymmetric unit contoured at 4σ and mapped on the final isomorphous refined model.The location of all 24 Cys or Met sulfur atoms, including disulfide bonds, were identified in the anomalous difference map, which proved to be instrumental for substructure determination and structure solution.Intriguing, the crystals used for S-SAD phasing grew from crystallization solutions containing sulfates, and an anomalous difference Fourier map peak at each binding site provided experimental confirmation of the sulfate ligand identity.b-e, Visualization of the sigma-weighted 2Fo-Fc electron density map, shown at 1.0 RMSD contour level after the final round of refinement in phenix.refinefor the Sema5ATSR3-4-NO3, Sema5ATSR3-4-SO4, Sema5ATSR3-4-SOS, Sema5ATSR3-4 apo structures, respectively.The Sema5ATSR3-4-NO3 structure contained several nitrate ions, not just at the active site, due to the high, 6 M nitrate concentration in the crystallization solution.Sema5ATSR3-4-SO4 contains a single sulfate ion at both active sites of the Sema5ATSR3-4 dimer.Sema5ATSR3-4-SOS contains a single SOS molecule per Sema5ATSR3-4 dimer possibly due to limitations imposed by the crystal packing.f, Trp-ladder of TSR3 is stabilized by mannosyl-Trp/Arg H-bonds, visualized from the Sema5ATSR3-4-NO3 structure.We identified well resolved mannosyl residues in 1 C4 chair conformation with a C-glycosidic attachment of C1 of an α-mannose to the indole C2 of W656 and W659 residues.The hydroxyl groups of these mannosyl moieties engage in a hydrogen-bond network to stabilize the sidechain orientations within the Trp-Arg ladder, similarly to what was shown for BaI1 1 and Unc-5 2 , which is expected to assist protein folding and secretion 3 .Residues are shown as sticks and the refined 2Fo-Fc electron density map is shown at 1.0 RMSD contour level.H-bonds are indicated as dashed lines.R676 that is affected by a deleterious missense ASD related mutation p.R676C 4 is highlighted in red letters.g, Anti-His Western blot analysis of Sema5Asema-TSR1-7-His6 (WT) and Sema5Asema-TSR1-7-His6 R676C (R676C) small-scale protein expression, performed as described in 5 .Media of transiently transfected HEK293T cells 3 days post-transfection were loaded on SDS-PAGE under reducing conditions.M1: Benchmark Ladder (His-tagged proteins), M2: Benchmark Prestained Ladder (non-His-tagged).Source data are provided as a Source Data file.Supplementary Figure 6.Sequence conservation of Sema5A TSR domains a, Sequence comparison of the human TSR1-7 domains.Sema5A residues that contribute to form either disulfide bonds, Trp-Arg ladder, or GAG site are indicated below the sequences.The Trp-Arg ladder is conserved in all 7 TSR repeats.TSR repeats no.2,3,5,6,7 include W-x-x-W-xx-W-x-x-C sequence motif that was identified as a canonical C-mannosylation motif for DPY19L1 9 .Among disulfide bridges that lock the β-strands of the TSR fold together (cf.Fig. 1f) #1 (pink) and #3 (blue) are conserved in all TSRs except TSR4 whereas TSRs 2,3,5,6 also feature disulfide bridge #2 (cyan).Note that no. 4 and 5 disulfide bonds covalently link TSR3 and 4 domains, respectively, from different monomers.The disulfide bridge conservation across TSR domains further indicates that the 3D domain swapped topology of TSR4, that is required for GAG site formation, is not shared with other TSRs within Sema5A.There is also no sequence conservation for the positively charged residues that constitute the GAG binding site of TSR4.b, TSR 3-4 domains are highly conserved within vertebrate class 5 semaphorins.Structure-based sequence alignment of the TSR 3-4 domain region from class 5 semaphorins.
Sequence numbering corresponds to human Sema5A.Domain regions and secondary structure elements of Sema5ATSR3-4 are shown above the sequence with 310 helix and beta strands represented as helix and arrows, respectively.Red shaded boxes enclose conserved positions and red letters show positions with homologous residues.Sema5A residues that form either disulfide bonds, Trp-Arg ladder and GAG site are indicated below the sequences.Note that no. 4 and 5 disulfide bonds covalently link TSR3 and 4 domains, respectively, from different monomers.Members of the semaphorin-5 class and their UniProt IDs (in order) in this alignment include: human Sema5A and Sema5B, Q13591 and Q9P283; Macaca mulatta (Rhesus macaque, MACMU) Sema5A and Sema5B, I2CT92 and NCBI RefSeq ID: XP_014985494.2;mouse Sema5A and Sema5B, Q62217 and Q60519; chicken Sema5A and Sema5B (CHICK), R4GFU3 and F1NSD7; Lacerta agilis (sand lizard, LACAG) Sema5A and Sema5B, NCBI RefSeq IDs XP_033009991.1 and XP_033018485.1;Danio rerio Sema5A and Sema5B (zebrafish, DANRE), F1R9L1 and F1QPQ6; fruit fly Sema5C, (DROME), Q9U631.Sequences were aligned using Clustal Omega and formatted using ESPript.Supplementary Figure 7. Ligand interactions and packing analysis of Sema5A crystal structures.
Schematic representation of ligand interactions at the GAG site of Sema5ATSR3-4 is shown for cocomplexed structures with a, sulfate, b, nitrate and c, sucrose octasulfate (SOS) within the crystal asymmetry unit, adapted from LigPlot +10 .Potential hydrogen bonds are shown as dashes.Note that interactions for only nitrates that are coordinated at the GAG site are shown here.d, Schematic representation of interactions of a single SOS molecule with residues from three crystallographic copies of Sema5ATSR3-4 dimers, adapted from LigPlot + .Chain labels are shown in parentheses for each residue, with A&B, C&D and E&F each referring to a Sema5ATSR3-4 dimer, respectively.SOS form additional interactions with Sema5ATSR3-4 beyond the core GAG binding site that accommodates sulfate and nitrate ligands.Some of these contacts may be considered for providing auxiliary interactions to further improve binding affinity and specificity to a GAG interaction partner of Sema5A.e-g, Polder mFobs-DFmodel omit maps (green) contoured at 2.5 σ on crystallographic ligands in e, Sema5ATSR3-4-SO4 f, Sema5ATSR3-4-NO3 g, Sema5ATSR3-4-SOS structures.h, A Sema5ATSR3-4 dimer together with its two crystallographic copies coordinate together a single SOS molecule.i, A single Sema5ATSR3-4 dimer forms crystal lattice contacts with three SOS molecules.Note that SOS molecules line up at binding site '1' while ligand binding at the opposite binding site '2' is precluded due to steric hindrance from crystal packing.Supplementary Figure 8. Analysis of Sema5A in silico docking with heparin tetrasaccharides Close-up structural views of each docked IdoA(2S)-GlcNS(6S)-IdoA(2S)-GlcNS(6S) tetramer heparin oligosaccharide poses, highlighting protein residues that are forming hydrogen bonds with the ligand.ClusPro clusters retrieved 1,000 models using interface RMSD with a 9-Å radius and ranks the clusters based on cluster population.List of the clusters and their energy scores are shown.
a, Image of agarose gel showing Ethidium bromide stained PCR products amplified from Sema5a GAG/+ , Sema5a GAG/GAG , Sema5a +/+ , and Sema5a GAG/GAG genomic DNA.The molecular weight of the PCR products is 293 bp.b, Western blot analysis of forebrain homogenates prepared from Sema5a +/+ , Sema5a GAG/+ , and Sema5a GAG/GAG P7 pups.WGA pull-down (WGA) of glycoproteins from brain lysates; Lysate: whole brain lysate before WGA pull-down.Blots were probed with anti-Sema5A or anti-actin to control for protein loading.c-e Sagittal sections of 3-month-old mouse brains stained with fluoromyelin (green) and DAPI (blue).Rostral is toward the left side.The habenula (Hb) in the dorsal thalamus and the fasciculus retroflexus (FR) are labeled.No obvious defects in FR fasciculation, thickness, or projection toward the interpeduncular nucleus in the midbrain were observed.Representative images of c wild-type, d Sema5a-/-, and e Sema5a GAG/GAG brains are shown (n= 3 per genotype).Scale bar, 500 µm.f, g Binding of recombinant alkaline phosphatase tagged Sema5A thrombospondin repeats 1-4 (AP-Sema5A-TSR1-4) harboring wildtype (WT) or GAG binding deficient (K734E,R747E,R749E, cf.Supplementary Fig. 1d) (G151) TSRs to coronal sections of the P0 mouse head.WT TSR1-4 binds strongly and broadly to tissue sections, including the developing hippocampus (arrow in f), and the inner retina (arrow in g).Scale bar, 1000 µm.Source data are provided as a Source Data file.
a, Dot plots analysis of snRNAseq dataset of WT P10 with marker genes used for cell type identification.b, Dot plots analysis of genes of interest revels cell type specific distribution and expression levels.Gene expression levels are normalized to average gene expression (color coded calibration).For each cell cluster, the percentile of cells expressing a specific gene product is indicated by the dot size.The snRNAseq dataset (GEO: GSE186216, 13 ) was re-analyzed.Astrocytes (Astro), neural stem cells (NSC), ependymal cells (EP), immature granulate cells (iGC), mature granule cells (mGC), pyramidal neuron (Py), Cornu Ammonis (CA), excitatory neurons, non-pyramidal (exNeuron), interneuron (IN), Cajal-Retzius cells (CR), presubicular neurons (PS), subicular neurons (SN), oligodendrocyte progenitor cell (OPC), microglia (MG), endothelial cells (EC), pericytes (Peri), mesenchymal cells (Mes), unknown cluster (UK).significance in progenitor cell migration but not in the regulation of dendritic spine density.We provide evidence that GAG-binding-deficient Sema5A functions as a null allele in migrating granule cells.We propose that multimerization of Sema5A on the surface of migrating progenitor cells influences adhesive and repulsive interactions, molecular events important for directed cell migration.Illustration is created with BioRender.com.

SupplementaryFigure. 3 .
Inter-domain hinge in Sema5ATSR3-4 Monomer chains from Sema5ATSR3-4-SO4 and Sema5ATSR3-4-NO3 co-crystal structures, abbreviated here as SO4-A/B or B or NO3-A/B, respectively, were superposed based on the TSR3 domain by PDBeFOLD6 .Domain translation, rotation and Cα atom root-mean-square deviation (RMSD) values were calculated in Pymol using the draw_rotation_axis.pyscript to compare Sema5ATSR3-4 chains within and across crystal forms.The C-terminal epitope tag peptide ( 766 GTLEVLFQ 773 ) remaining after HRV 3C protease cleavage was neither included into the superimpositions nor visualized here.

Supplementary Figure 4 .
Structural homologs of the Sema5A TSR3 domain.Structural homology search for individual Sema5A TSR3 (Sema5ATSR3-4-NO3 chain residues 653-702) and TSR4 (Sema5ATSR3-4-NO3 chain A residues 703-754) domains was carried out using the DALI server 7 against the PDB50 database.Structure comparison data with the ten closest structural homologs of Sema5A TSR3 are shown.a,-e, Pairwise structural superimpositions of Sema5A TSR3 with the top five structural homolog domains from unrelated proteins.Disulfides stabilizing the TSR fold are shown as sticks.f, Structural superimposition of Sema5A TSR3 and TSR4 domains.Note that no structural homologs were retrieved for the TSR4 DALI50 query.Supplementary Figure 5. Sema5ATSR3-4 homodimer interactions.a, and b, schematics of the Sema5A TSR3 and TSR4 homodimer interactions adapted from PDBSUM 8 , based on the Sema5ATSR3-4-NO3 structure.The buried surface area of the respective protein chain is shown in brackets in a. Residue colours code in b: Positive (H,K,R); negative (D,E); S,T,N,Q = neutral; A,V,L,I,M = aliphatic; F,Y,W = aromatic; P,G = Pro&Gly; C = Cysteine.c, Cartoon presentation of Sema5ATSR3-4 with close-up views in d-f, displaying key inter-chain interactions that together with the β-sheet hydrogen bond network stabilizes the dimer architecture.Colour coding as in Fig. 1.
Values in parentheses correspond to the highest resolution shell unless otherwise stated.RMSD: Root Mean Square Deviations.Asterisks (*) denote reciprocal space.